Rapid and Efficient CRISPR/Cas9-Based Mating-Type Switching of <i>Saccharomyces cerevisiae</i>

Abstract

Rapid and highly efficient mating-type switching of <i>Saccharomyces cerevisiae</i> enables a wide variety of genetic manipulations, such as the construction of strains, for instance, isogenic haploid pairs of both mating-types, diploids and polyploids. We used the CRISPR/Cas9 system to generate a double-strand break at the <i>MAT</i> locus and, in a single cotransformation, both haploid and diploid cells were switched to the specified mating-type at ∼80% efficiency. The mating-type of strains carrying either rod or ring chromosome III were switched, including those lacking <i>HML</i>α and <i>HMR</i> <b>a</b> cryptic mating loci. Furthermore, we transplanted the synthetic yeast chromosome V to build a haploid polysynthetic chromosome strain by using this method together with an endoreduplication intercross strategy. The CRISPR/Cas9 mating-type switching method will be useful in building the complete synthetic yeast (Sc2.0) genome. Importantly, it is a generally useful method to build polyploids of a defined genotype and generally expedites strain construction, for example, in the construction of fully <b>a/a</b>/α/α isogenic tetraploids.